1. Field of the Invention
The present invention relates to the field of immunology and animal models, and, more particularly, to non-human chimeric mammals, where hematopoietic cells have been replaced by hematopoietic cells received from donor mammals having a hematopoietic deficiency, and to methods for their production, wherein such chimeric animals may be used as animal models of bacterial toxin shock.
2. Description of the Background Art
Bacteria produce endotoxins and exotoxins which are known to cause toxin related pathologies, such as toxic shock in humans, which can result in death. Exotoxins are usually heat-labile proteins of great toxicity, typically formed by gram-positive bacteria, e.g. of the genera Micrococcus, Staphylococcus, Streptococcus, Corynebacterium, Lactobacillus, Bacillus and Clostridium. The endotoxins are usually heat-stable lipopolysaccharide-protein complexes of high toxicity, typically formed by gram-negative bacteria, e.g., of the genera Brucella, Haemophilus, Escherichia, Klebsiella, Proteus, Salmonella, Pseudomonas, Shigella, Vibrio, Yersinia, etc. Staphylococcus aureus, in particular, produces an exotoxin designated Staphylococcal enterotoxin B (SEB), that has been associated with toxic shock syndrome (TSS), a syndrome characterized by high fever, vomiting, diarrhea, confusion, and skin rash that may rapidly progress to severe and intractable shock. Septic shock, a disease with significant morbidity and with overall mortality from 50 to 90%, is associated with bacteremia due to gram-negative bacteria or meningococci.
Research in the bacterial shock field has historically been hindered by the absence of relevant and cost-effective animals models. Laboratory animals, including severe combined immune deficient (SCID) mice and other mutant mice, are highly insensitive to bacterial toxins. Models in other animals, including primates, are being developed but have not become widely used. Sensitivity to bacterial toxins was found to be increased in mice by inducing liver toxicity with i.p. injections of D-galactosamine (Galanos et al., Proc. Natl. Acad. Sci. U.S.A. 76:5939 (1979); Lehmann et al., J. Exp. Med. 165:657 (1987)).
The mode of action of bacterial exotoxins is unclear as yet. Some of these toxins exhibit the unusual properties of superantigens, and are able to bind to human and mouse major class II histocompatibility antigens and the complex formed can stimulate a large number of T cells, expressing appropriate Vxcex2 segments of the T cell receptor (TCR). It is likely that this ability to induce a massive T cell proliferation is related, at least in part, to the pathological effects of the toxins, since it was shown that T cell-derived lymphokines appear to cause shock-like syndromes and that T cell repopulation of SCID mice conferred sensitivity to Staphylococcal enterotoxin B (SEB). However, because mice are more resistant than humans to the pathogenic effects of bacterial toxins, the latter experiment was performed in a mouse model in which liver metabolism was impaired by i.p. injection of D-galactosamine (Miethke et al., J. Exp. Med. 175:91-98 (1992)). Although this D-galactosamine sensitized mouse model provides some increase in sensitivity to SEB, the other physiological effects of D-galactosamine may be expected to make such a mouse model unsuitable as the response is not shown to directly mimic the physiological effects found in human SEB shock.
Wood et al., FEMS Microbiology Immunology 76: 121-134 (1991), disclose that in animal models, humans and primates react similarly to staphylococcus enterotoxins (SEs) and, for this reason, the monkey has been used to study the effects of SE""s following either oral or intra-venous administration. However, primates are not the ideal model and it has proved difficult to find a suitable alternative. Dwarf goats and rabbits are susceptible to SE""s, but neither is appropriate for large-scale studies. Rodents are relatively unreactive, being capable of surviving large doses of toxin.
The lack of similar response in animal models, such as mice or other rodents, has hindered the development of a suitable model of human bacterial toxin related pathologies. Accordingly, there is a need to provide chimeric non-human mammals for use as animal models for human bacterial toxin shock by providing animal models that respond similarly to humans in terms of symptomology, pathology and therapeutic response.
EP 469,632, published Feb. 5, 1992, by McCune et al. and assigned to SyStemix, Inc., discloses the determination of the response of a pathogen (other than retroviruses, such as bacteria) tropic to a particular species, to a stimulus by (1) administration of a stimulus, able to induce a physiological response from tissue cells of the particular species, to an immunocompromised host, other than a primate, lacking at least functional T cells; and (2) determining the effect of the stimulus, wherein the tissue cells are vascularized, non-transformed solid organ tissue. Also SCID/hu mice (having human transplanted tissue) are disclosed which are infected in the human tissue with a pathogen tropic for this tissue. The solid tissue is a hematopoietic tissue, and the host is a SCID/SCID mouse (having autosomal recessive mutation SCID). The SCID/hu mice can be used to evaluate the effect of drugs and vaccines, and of agents (or conditions) on specific tissues or on the immune system. The human tissue is disclosed to remain viable for more than four weeks.
Bosma et al. (Nature 301:527-530(1983)) describe and characterize an immunodeficient mouse as an autosomal recessive trait wherein differentiation of both B and T lymphocytes is impaired. To determine whether the particular C.B.-17 scid mice had a defective cellular environment for lymphoid differentiation, reciprocal bone marrow transfers were made between the SCID C.B.-17 scid mice and BALB/c mice. However, while the bone marrow from the BALB/c mice donors to SCID recipients showed the BALB/c bone marrow cell allotype, none of the SCID donor bone marrow allotype was found after transplant into BALB/c recipient. Accordingly, SCID bone marrow was not shown to survive in sublethally irradiated BALB/c mice, due to replacement by the BALB/c irradiated bone marrow cells, such that transplant of SCID bone marrow into BALB/c mice is disclosed to be unsuccessful.
The use of T cell-deficient SCID mice as bone marrow cell donors in an allogeneic bone marrow transplantation model in which the recipient mice have destroyed bone marrow, resulted in markedly enhanced rejection compared to syngeneic control grafts (Murphy et al., J. Immunol. 144:3305-3331 (1990)). Accordingly, this reference discloses the unsuitability and lack of successful transplant of bone marrow cells into allogeneic or xenogeneic recipients having a destroyed immune system.
Citation of documents herein is not intended as an admission that any of the documents cited herein is pertinent prior art, or an admission that the cited documents are considered material to the patentability of the claims of the present application. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.
It is an object of the present invention to overcome one or more deficiencies of the related art.
For production of a useful non-human mammal model of human bacterial toxin pathologies according to the present invention, it has unexpectedly been discovered that non-human mammals, preferably rodents, normally resistant to bacterial toxins, are made susceptible to bacterial toxin pathologies present in humans, when their hematopoietic cells have been substantially destroyed and replaced by hematopoietic cells from non-human mammals having a hematopoietic deficiency. In a preferred embodiment, the recipient mammal is a mouse, more preferably mouse strain C3H/HeJ.
Thus, according to the present invention, an animal model for human bacterial toxin pathologies is provided by using a normal or other suitable non-human mammal M1, wherein the hematopoietic cells of the mammal M1 are substantially destroyed and replaced by hematopoietic cells derived from a non-human mammal M2 having a hematopoietic deficiency, to provide a chimeric non-human mammal M3, which is unexpectedly found to provide a non-human mammal model of human bacterial toxin-related pathologies.
The present invention is thus directed, in one aspect, to a non-human chimeric mammal M3 having long-term stable donor-type hematopoietic cells, comprising a mammal M1, the hematopoietic cells of which have been substantially suppressed or destroyed and replaced by hematopoietic cells originating from a mammal M2 having a hematopoietic deficiency, which mammal M3 is susceptible to bacterial toxin-related pathologies found in humans.
According to the present invention, any non-human mammal with a hematopoietic deficiency may be used as the donor, M2. The hematopoietic deficiency of M2 may be selected from severe combined immunodeficiency (SCID), an erythroid cell deficiency, a myeloid cell deficiency, a T cell deficiency, a B cell deficiency or any other hematopoietic deficiency. Useful mouse strains with hematopoietic deficiencies include immunodeficient SCID, Bg/Nu/Xid (BNX), Nu mice or erythroid deficient W/WV mice, or any combination thereof. Gene-deleted hematopoietic deficient mammals are also intended within the scope of the present invention as M2 donors.
The mammal donor M2 is of a species the same or other than the mammal recipient M1. Preferably, M1 and is M2 belong to the same rodent genus or species In preferred embodiments, M2 is a SCID mouse and M1 is a C3H/HeJ mouse.
The source of hematopoietic cells of a hematopoietic deficient mammal M2 used according to the present invention may be selected or derived from:
(a) unfractionated or fractionated bone marrow cells;
(b) unfractionated or fractionated blood cells;
(c) unfractionated or fractionated spleen or thymus cells;
(d) unfractionated or fractionated cord blood cells;
(e) unfractionated or fractionated fetal tissue (liver, thymus, bone marrow, spleen or blood); and
(f) any combination thereof.
The cells may be used directly, as cell cultures and/or after being passaged one or more times in vitro or in vivo, such as 2, 3, 4, 5 or more times.
The present invention also provides a method for the production of a non-human chimeric mammal M3, comprising:
a) treating a non-human mammal M1 so as to substantially destroy the immune system of M1; and
b) transplanting the treated mammal M1 with hematopoietic cells originating from a mammal M2 having a hematopoietic deficiency;
thereby obtaining a chimeric non-human mammal M3 having hematopoietic cells derived from mammal donor M2, wherein M3 is susceptible to bacterial toxin shock or other bacterial toxin-related pathological conditions such as those found in humans.
The non-human chimeric M3 mammals of the present invention can be used as animal models to test and evaluate drugs and/or diagnostic agents for human pathologic conditions involving bacterial toxins, wherein the mammal M3 responds to bacteria or at least one toxin as administered as an intramuscular, intravenous or subcutaneous injection, or via ingestion, aerosol, inhalation, or other respiratory, topical or gastrointestinal introduction or by exposure to the bacterial toxin or bacteria in a similar physiological manner as humans may be exposed
The invention also comprises a method for evaluating a human bacterial toxin pathology in a non-human animal model, comprising:
a) administering to a non-human chimeric mammal M3 as described herein with a bacterial toxin; and
b) evaluating the physiological response of administered mammal M3, as a model of human bacterial toxin shock.
The invention further comprises a method for evaluating potential therapeutic agents for the treatment of human bacterial toxin shock in a non-human animal model, comprising:
a) administering to a non-human chimeric mammal M3 as described herein with a bacterial toxin;
b) administering said therapeutic agent in a therapeutically effective amount to said chimeric mammal M3; and
c) evaluating the physiological response of administered mammal M3, as a model of human bacterial toxic shock to determine the effectiveness of said therapeutic agent in treating or preventing the pathological effects of exposure to said bacterial toxin,
wherein steps (a) and (b) can be performed concurrently, (a) before (b) or (b) before (a).